Steroid regulation plays a central role in the health and development of adults and children. Disrupted or altered regulation of steroids is associated with a wide host of diseases, including Addison Disease, congenital hyperplasia, Cushing's disease, Hirsutism, Conn's disease, polycystic ovary disease and gynecomastia, among others. Often disease states will differ based on subtle variations in the amount of or the complex series of interactions among the many different steroids. Differential diagnosis, therefore, depends on the simultaneous and quantitative analysis of multiple steroids.
Steroid biosynthesis is a complex metabolic pathway utilizing simple precursors to synthesize multiple steroidal forms. This biosynthetic pathway that is unique to animals provides a common target for antibiotics and other anti-infective drugs. In addition, synthetic steroids and steroid derivatives are frequently used in therapeutic applications. Many of the steroid structures are closely related making their analysis challenging even when using the selectivity of mass spectrometric detection. Chromatographic separation is therefore essential for accurate and precise analysis of clinically relevant steroids and steroid derivatives.
Typical methods for analysis of steroids and steroid derivatives include Immunoassay (IA), GC/MS, and LC/MS/MS. However, there are shortcomings associated with each of these methods. For example, the IA methods are non-specific, time-consuming, labor-intensive and do not lend themselves well to rapid or high throughput analyses of multiple analytes or samples. The gas chromatography/mass spectroscopy (GC/MS) methods require sample samples. The gas chromatography/mass spectroscopy (GC/MS) methods require sample derivitization prior to GC analysis, which is burdensome and time-consuming. In LC/MS/MS methods, although no sample derivitization is required, the typical run time of a sample on an HPLC (high performance or pressure liquid chromatography) instrument is about 12 minutes; which has recently been reduced to about 4-5 minutes by using a UHPLC (ultra high performance or pressure chromatography) instrument. However, there are several disadvantages to using HPLC or UHPLC, one of which being their using of toxic organic solvents as mobile phase and generating excess toxic waste, which is expensive to purchase and dispose of.
The use of non-toxic Supercritical CO2 (SC—CO2) as an alternative to organic solvents as the mobile phase has resulted in the advent of supercritical fluid chromatography (SFC) which embraces many of the features of liquid and gas chromatography. Theoretically, SC—CO2 provides a low viscosity mobile phase that achieves higher diffusion rates and enhanced mass transfer over the solvents used in HPLC. However, the current SFC instruments (which are mainly retooled HPLCs) and methods have many limitations including, for example, long sample run time, inaccurate or imprecise control over the mobile phase density and composition, inability to reliably modifiers at low amounts (<5% per volume of CO2), susceptibility to system pressure fluctuations and sample backflow, baseline noise, sample carryover, and lack of robustness, which prevent users from rapidly obtaining reproducible results.
Therefore, there still remains a need for an improved chromatography system and method that overcomes one or more of the above limitations and allow for a rapid and robust analysis of steroids and steroid derivatives.